Biosynthesis of Phosphoserine in the Methanococcales

Abstract

Methanococcus maripaludis and Methanocaldococcus jannaschii produce cysteine for protein synthesis using a tRNA-dependent pathway. These methanogens charge tRNA Cys with L-phosphoserine, which is also an intermediate in the predicted pathways for serine and cystathionine biosynthesis. To establish the mode of phosphoserine production in Methanococcales, cell extracts of M. maripaludis were shown to have phosphoglycerate dehydrogenase and phosphoserine aminotransferase activities. The heterologously expressed and purified phosphoglycerate dehydrogenase from M. maripaludis had enzymological properties similar to those of its bacterial homologs but was poorly inhibited by serine. While bacterial enzymes are inhibited by micromolar concentrations of serine bound to an allosteric site, the low sensitivity of the archaeal protein to serine is consistent with phosphoserine's position as a branch point in several pathways. A broad-specificity class V aspartate aminotransferase from M. jannaschii converted the phosphohydroxypyruvate product to phosphoserine. This enzyme catalyzed the transamination of aspartate, glutamate, phosphoserine, alanine, and cysteate. The M. maripaludis homolog complemented a serC mutation in the Escherichia coli phosphoserine aminotransferase. All methanogenic archaea apparently share this pathway, providing sufficient phosphoserine for the tRNA-dependent cysteine biosynthetic pathway.Many methanogenic archaea have a remarkable tRNA-dependent pathway for cysteine biosynthesis. In these organisms, an unusual class II aminoacyl-tRNA synthetase catalyzes the aminoacylation of tRNA Cys with L-phosphoserine (Sep) (38). This Sep-tRNA Cys is then sulfurylated to produce Cys-tRNA Cys for protein biosynthesis. This tRNA-dependent pathway for cysteine biosynthesis conserves the energy of a phosphoester bond compared to the canonical pathway, where phosphoserine is hydrolyzed to produce serine that is acetylated and then converted to cysteine by a sulfhydrolase. Methanocaldococcus jannaschii also uses phosphoserine to produce cystathionine (50). This paper addresses the biosynthesis of phosphoserine in Methanococcus maripaludis and M. jannaschii to decipher the relationship between cysteine and serine biosynthesis in the euryarchaea. Three pathways for the biosynthesis of serine are the phosphorylated pathway, which produces a phosphoserine intermediate (16, 18), the nonphosphorylated pathway (36), and the serine pathway for incorporating formaldehyde by the reverse activity of serine hydroxymethyltransferase (34). Most microorganisms use the phosphorylated pathway, which requires three dedicated enzymes, namely, phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase, and phosphoserine phosphatase. Many eukaryotes and some bacteria employ the nonphosphorylated pathway, which requires phosphoglycerate kinase, glycerate dehydrogenase, and serinepyruvate aminotransferase. The serine pathway for C 1 assimilation is commonly found in methanotrophic and methylotrophic bacteria.Isotopic labeling...

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